Our long-term goal is to define the molecular target(s) of ethanol that explain the neurophysiological basis of intoxication. Several lines of evidence suggest that modulation of the gamma-aminobutryic acid (GABA) neurotransmitter receptor is tat least partially responsible for mediating the acute intoxicating effects of ethanol. To further understand the fuunctional significance of this receptor as it pertains to the mechanism of ethanol intexocation, we propose to use gene targeting in mouse embryonic stem cells to create two novel mouse lines, each containing a defined mutation in the gene encoding the gamma2 subtype of the GABA type A receptor (GABA/A-R). The mouse lines created will be used to test the hypothesis that the gamma2 subtype of the GABA/A-R is critically important to ethanol's intoxicating effects on animals. Both behavioral (loss-of- righting reflex) and subcellular-level (permeability and electrophysiologic) assays will be performed on mutant and wild type mice. The strategic advantage inherent in this proposal is that differences in response to ethanol will be directly linked to single, unambiguous molecular lesions in the gamma2 subtype gene of the GABA/A-R. In this revised application, we describe substantial Preliminary Data (that has been made) at cloning the genomic locus that encodes the gamma2 subtype of the GANA/A-R, at creating targeting constructs, at targeting other loci in embryonic stem cells, at using embryonic stem cells to create chimeric mice, at demonstrating the effects of ethanol on our 36/Cl-influx assay, and trichloroethanol on our electrophysiological assays, and at demonstrating the effects of ethanol on mice in our loss-of-righting reflex assay.